The proteasome is a multicatalytic proteolytic enzyme that is essential for the degradation of intracellular proteins thereby regulating processes such as signal-transduction, cell-cycle control, antigen processing and apoptosis. Inhibition of the proteasome offers a promising approach in the treatment of cancer. The peptide boronic acid bortezomib, for example, was FDA-approved for the treatment of multiple myeloma in 2003. Recently emerging side effects of bortezomib, however, have renewed the challenge to develop novel proteasome inhibitors. The majority of the inhibitors reported in the literature react with te catalytic threonine residue within the active site. The natural product TMC-95A, however, is a macrocyclic peptide that reversibly inhibits the proteasome through a hydrogen-bonding network between the enzyme subsites and the extended peptide backbone of the macrocycle. This proposal describes the design and synthetic strategy of novel macrocyclic peptidyl inhibitors that mimic the oxindole containing TMC-95 macrocycle. Both covalently and non-covalently binding inhibitors featuring this design are proposed. Three specific aims will explore these modifications. In Specific Aim 1 non-covalently binding TMC-95 analogs will be investigated. In Specific Aim 2 an electrophilic trap will be incorporated to compare covalently binding TMC-95 analogs to non-covalently binding analogs. In Specific Aim 3 enzyme and cellular assays and evaluation of physicochemical properties will determine the biological efficacy of the proposed inhibitors. These compounds offer a new and unique approach to effectively inhibit the proteasome, potentially leading to the development of new chemotherapeutic agents.